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EP3124118B1 - Mikrokanalvorrichtung und pcr-verfahren - Google Patents

Mikrokanalvorrichtung und pcr-verfahren Download PDF

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Publication number
EP3124118B1
EP3124118B1 EP16170761.7A EP16170761A EP3124118B1 EP 3124118 B1 EP3124118 B1 EP 3124118B1 EP 16170761 A EP16170761 A EP 16170761A EP 3124118 B1 EP3124118 B1 EP 3124118B1
Authority
EP
European Patent Office
Prior art keywords
microchannel
substrate layer
metal film
heating
temperature regulator
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Not-in-force
Application number
EP16170761.7A
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English (en)
French (fr)
Other versions
EP3124118A1 (de
Inventor
Mitsuoki Hishida
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Panasonic Intellectual Property Management Co Ltd
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Panasonic Intellectual Property Management Co Ltd
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Publication of EP3124118A1 publication Critical patent/EP3124118A1/de
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Publication of EP3124118B1 publication Critical patent/EP3124118B1/de
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0874—Three dimensional network
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves

Definitions

  • the present disclosure relates to a microchannel chip, a polymerase chain reaction (PCR) method, and a heating/cooling control apparatus, each of which is used in performing a PCR process on a treatment target liquid.
  • PCR polymerase chain reaction
  • PTL1 discloses a heating-reaction use microchip that includes a reaction region for various reactions to occur, and a heating control field that has a heat generating element and a liquid and controls heating of the reaction region.
  • the invention relates to a PCR method and microchannel apparatus as claimed in claims 1 and 8, with which the responsiveness of the temperature of a treatment target liquid undergoing a PCR process can be improved using a temperature regulator.
  • the techniques disclosed here feature a polymerase chain reaction (PCR) method for performing a PCR process on a treatment target liquid using a microchannel apparatus including a microchannel chip and a heating/cooling control apparatus, the PCR method including:
  • diffusion of heat of the temperature regulator can be reduced, and the temperature regulator can improve the responsiveness of the temperature of the treatment target liquid.
  • a first aspect of the present disclosure provides a polymerase chain reaction (PCR) method for performing a PCR process on a treatment target liquid using a microchannel apparatus including a microchannel chip and a heating/cooling control apparatus, the PCR method including:
  • diffusion of heat of the temperature regulator can be reduced, and the temperature regulator can improve the responsiveness of the temperature of the treatment target liquid.
  • a second aspect of the present disclosure provides the PCR method according to the first aspect, wherein the microchannel chip further includes:
  • the structure that prevents leakage of the liquid during the PCR process is advantageously obtained.
  • a third aspect of the present disclosure provides the PCR method according to one of the first and second aspects, wherein the second substrate layer body is made of dimethylpolysiloxane, and the metal film is made of aluminum.
  • use of aluminum being higher than dimethylpolysiloxane in thermal conductivity advantageously allows the temperature regulator to quickly heat or cool.
  • a fourth aspect of the present disclosure provides the PCR method according to any one of the first to third aspects, wherein the temperature regulator is a Peltier element.
  • the Peltier element can raise or lower the temperature to any value and conduct the heat to the metal film, thereby achieving the PCR process.
  • a fifth aspect of the present disclosure provides the PCR method according to any one of the first to fourth aspects, wherein the metal film includes a plurality of metal films that are spaced apart from each other and arranged along the microchannel.
  • the temperature regulator can be efficiently brought into contact with the metal film.
  • the efficient heating and cooling process is realized.
  • a sixth aspect of the present disclosure provides the PCR method according to the second aspect, further including, after the step (b), (d) opening the introduction-side valve and the discharge-side valve so as to discharge, from the discharge-side valve, the liquid having undergone the PCR process.
  • the structure that prevents leakage of the liquid during the PCR process is advantageously obtained, and the liquid can be taken out at any timing.
  • a seventh aspect of the present disclosure provides the PCR method according to any one of the first to sixth aspects, wherein, in the step (b), the heating/cooling controller controls the change in the temperature of the temperature regulator in accordance with a type of the liquid, based on relationship information among the type of the liquid, the voltage to the temperature regulator, a period of applying the voltage, and the temperature.
  • the heating or cooling period can be efficiently controlled.
  • An eighth aspect of the present disclosure provides a microchannel apparatus according to claim 8.
  • diffusion of heat of the temperature regulator can be reduced, and the temperature regulator can improve the responsiveness of the temperature of the treatment target liquid.
  • a ninth aspect of the present disclosure provides the microchannel chip according to the eighth aspect, wherein the microchannel chip includes:
  • the flow of the liquid can be blocked for any period by causing the introduction-side valve and the discharge-side valve to be pushed.
  • a tenth aspect of the present disclosure provides the microchannel chip according to one of the eighth and ninth aspects, wherein the second substrate layer body is made of dimethylpolysiloxane, and the metal film is made of aluminum.
  • the heat by the temperature regulator is not easily conducted to the second substrate layer body and tends to stay at the metal film.
  • an end of the metal film of the second substrate layer in the microchannel chip is buried in the second substrate layer body.
  • the effect of preventing leakage of the liquid can be achieved.
  • FIG. 1A is a perspective view of the overall structure of microchannel system 1, which is a microchannel apparatus according to one exemplary embodiment of the present disclosure.
  • FIGS. 1B and 1C are perspective views, as seen from above and below, respectively, of a microchannel portion of the microchannel apparatus according to the present exemplary embodiment.
  • Microchannel system 1 shown in FIGS. 1A to 1C includes microchannel chip 10 and heating/cooling control apparatus 20 being an exemplary heating/cooling control apparatus.
  • Microchannel chip 10 includes first substrate layer 100 and second substrate layer 200.
  • Heating/cooling control apparatus 20 includes temperature regulator 11, power source 12, and heating/cooling controller 13.
  • Microchannel system 1 amplifies DNA in treatment target liquid 50 in microchannel chip 10 through the polymerase chain reaction (PCR) method. Specifically, microchannel system 1 controls temperatures of treatment target liquid 50 in microchannel chip 10 through use of temperature regulator 11, power source 12, and heating/cooling controller 13. Treatment target liquid 50 contains a gene (i.e., DNA). Treatment target liquid 50 may be referred to as "Analyte”.
  • PCR polymerase chain reaction
  • FIGS. 2A to 2C are vertical cross-sectional views and a plan view of microchannel chip 10.
  • Microchannel chip 10 includes first substrate layer 100 and second substrate layer 200.
  • first substrate layer 100 is structured by first substrate layer body 100a that includes introducing channel 101 and discharging channel 102, and back plate 100b that is fixed to the lower surface of first substrate layer 100.
  • FIG. 1A shows the bottom surface of channels 101, 102 of first substrate layer 100 being structured by back plate 100b, back plate 100b can be dispensed with if introducing channel 101 and discharging channel 102 are structured inside first substrate layer body 100a.
  • first substrate layer body 100a examples include dimethylpolysiloxane (PDMS), polycarbonate, acrylic resin (polymethyl methacrylate (PMMA)), and silicone.
  • exemplary materials of back plate 100b include dimethylpolysiloxane, polycarbonate, acrylic resin, silicone, and glass.
  • Introducing channel 101 includes first channel 101a, second channel 101b, and fifth channel 101d.
  • First channel 101a extends in parallel to the surface of first substrate layer 100 via introduction-side valve 51 which will be described later.
  • Second channel 101b is connected to the downstream end side of first channel 101a, and extends substantially perpendicularly to the surface of first substrate layer 100 (that is, toward second substrate layer 200), to penetrate through first substrate layer 100 in the thickness direction.
  • Fifth channel 101d is connected to the upstream end side of first channel 101a, and extends substantially perpendicularly to the surface of first substrate layer 100 (that is, toward second substrate layer 200), to penetrate through first substrate layer 100 in the thickness direction.
  • Discharging channel 102 includes third channel 102a, fourth channel 102b, and sixth channel 102d.
  • Third channel 102a extends in parallel to the surface of first substrate layer 100 via introduction-side valve 51 which will be described later.
  • Fourth channel 102b extends substantially perpendicularly to the surface of first substrate layer 100, to penetrate through first substrate layer 100 in the thickness direction.
  • Fifth channel 101d is connected to the upstream end side of first channel 101a, and extends substantially perpendicularly to the surface of first substrate layer 100, to penetrate through first substrate layer 100 in the thickness direction.
  • Second substrate layer 200 is disposed on first substrate layer 100.
  • Second substrate layer 200 includes at least second substrate layer body 200a that has microchannel 202 at its inner central portion, and metal film 200b that is fixed to the upper portion of the central portion of substrate layer body 200a so as to structure the upper surface of microchannel 202.
  • metal film 200b enters inside substrate layer body 200a, as if it is pressed down by an In such a structure, metal film 200b having high thermal conductivity is partially covered with the material of substrate layer body 200a. This increases the contact area between metal film 200b and substrate layer body 200a, minimizing leakage of liquid 50.
  • FIG. 1A and others show screw holes 200e for fixing second substrate layer 200 to first substrate layer 100.
  • Metal film 200b includes the outer surface that is brought into direct contact with temperature regulator 11, and the inner surface that is brought into direct contact with treatment target liquid 50.
  • An exemplary material of metal film 200b is aluminum having high thermal conductivity.
  • second substrate layer 200 is silicone-based resin.
  • silicone-based resin is dimethylpolysiloxane. That is, second substrate layer 200 being made of a low-thermal-conductivity material reduces diffusion of heat, which is conducted from temperature regulator 11 to metal film 200b, to substrate layer body 200a. Further, metal film 200b, which is the upper surface of second substrate layer 200 brought into direct contact with treatment target liquid 50, being made of high-thermal-conductivity metal allows temperature regulator 11 to control the temperature of treatment target liquid 50 via metal film 200b with ease.
  • Second substrate layer 200 may further integrally include introduction-side valve 51, discharge-side valve 52, introducing opening 101c, and discharging opening 102c.
  • Introduction-side valve 51 is integrally formed with second substrate layer 200 as a part of second substrate layer 200 around the intermediate portion of first channel 101a of introducing channel 101.
  • Introduction-side valve 51 is structured by valve body 51a, thin and elastically deformable supporters 51b that constantly apply biasing force to valve body 51a so as to be kept at open position I, and recess 51c that is formed at the bottom surface of valve body 51a. Opening ends of a pair of upward channels 101e at the intermediate portion of first channel 101a of introducing channel 101 face recess 51c. Accordingly, when valve body 51a is at the upper end position, i.e., open position I, by the biasing force of supporters 51b, upward channels 101e communicate with each other via recess 51c. Accordingly, for example, treatment target liquid 50 is allowed to be introduced from introducing opening 101c to microchannel 202 via first channel 101a and second channel 101b of introducing channel 101.
  • valve body 51a when valve body 51a is lowered against the biasing force of supporters 51b and positioned at close position II, the pair of upward channels 101e is closed by the elastically deformed bottom surface of recess 51c; first channel 101a is closed at the intermediate portion; and treatment target liquid 50 can be packed and retained in introducing channel 101 and microchannel 202. Opening and closing operations of introduction-side valve 51 in such a manner enables control over introduction and packing and retaining of liquid 50 flowing through first channel 101a.
  • Discharge-side valve 52 is integrally formed with second substrate layer 200 as a part of second substrate layer 200 around the intermediate portion of fourth channel 102b of discharging channel 102.
  • discharge-side valve 52 is structured by valve body 52a, thin and elastically deformable supporters 52b that constantly apply biasing force to valve body 52a so as to be kept at open position I, and recess 52c that is formed at the bottom surface of valve body 52a. Opening ends of a pair of upward channels 102e at the intermediate portion of fourth channel 102b of discharging channel 102 face recess 52c. Accordingly, when valve body 52a is at the upper end position, i.e., open position I, by the biasing force of supporters 52b, upward channels 102e communicate with each other via recess 52c.
  • treatment target liquid 50 is allowed to be discharged from microchannel 202 through discharging opening 102c via third channel 102a and fourth channel 102b of discharging channel 102.
  • valve body 52a is lowered against the biasing force of supporters 52b and positioned at close position II, the pair of upward channels 102e is closed by the elastically deformed bottom surface of recess 52c; fourth channel 102b is closed at the intermediate portion; and treatment target liquid 50 can be packed and retained in microchannel 202 and discharging channel 102. Opening and closing operations of discharge-side valve 52 in such a manner enables control over discharge of the liquid flowing through fourth channel 102b.
  • introduction-side valve 51 and discharge-side valve 52 may be normally kept at open position I by the biasing force of supporters 51b, 52b.
  • introduction-side valve 51 and discharge-side valve 52 may be closed so as to position at close position II against the biasing force of supporters 51b, 52b.
  • Microchannel 202 includes first end 202a that is connected to the downstream end of introducing channel 101, and second end 202b that is connected to the upstream end of discharging channel 102.
  • Microchannel 202 is formed to meander, for example, in an S-shaped manner, from first end 202a to second end 202b.
  • the inner surface of heating/cooling-purpose metal film 200b which is fixed to the upper portion of the central portion of second substrate layer 200 and has a circular shape, for example, is exposed.
  • microchannel 202 meanders in an S-shaped manner so that the liquid is brought into contact with the inner surface of metal film 200b to a maximum extent, and thus liquid 50 in microchannel 202 can be efficiently brought into contact with metal film 200b of a smaller area.
  • treatment target liquid 50 is introduced into first end 202a of microchannel 202.
  • the introduced treatment target liquid 50 meanderingly flows through microchannel 202 while being brought into contact with metal film 200b and thereby heated or cooled.
  • treatment target liquid 50 is discharged to the upstream end of discharging channel 102 at first substrate layer 100.
  • temperature regulator 11 is disposed so as to be in contact therewith.
  • heat of temperature regulator 11 can be efficiently conducted to treatment target liquid 50 via metal film 200b at the upper surface of second substrate layer 200.
  • introducing opening 101c is connected via introduction-side valve 51.
  • Introducing opening 101c is a recess having an inclined surface. Introducing opening 101c penetrates through second substrate layer 200 in the thickness direction, to be connected to the upstream end of fifth channel 101d.
  • treatment target liquid 50 is supplied to introducing opening 101c by a pump or a dropper. Then, treatment target liquid 50 enters first channel 101a from fifth channel 101d of introducing channel 101, and flows through first channel 101a to be introduced to microchannel 202 at second substrate layer 200 from second channel 101b.
  • discharging opening 102c is connected via discharge-side valve 52.
  • Discharging opening 102c is a recess having an inclined surface. Discharging opening 102c penetrates through second substrate layer 200 in the thickness direction, to be connected to the downstream end of sixth channel 102d. After a PCR process, treatment target liquid 50 is discharged from microchannel 202 at second substrate layer 200 to third channel 102a of discharging channel 102, and flows through fourth channel 102b, to be output from sixth channel 102d and discharging opening 102c to a DNA sensing apparatus (not shown), for example.
  • Heating/cooling control apparatus 20 includes housing 20a, temperature regulator 11 fixed to the central portion of the lower surface of housing 20a, power source 12, and heating/cooling controller 13. Heating/cooling control apparatus 20 may further include valve controller 104 on each of the opposite sides on lower surface of housing 20a.
  • Power source 12 and heating/cooling controller 13 may be disposed inside or outside housing 20a.
  • Temperature regulator 11 is fixed to the lower surface at the central portion of housing 20a of heating/cooling control apparatus 20. Valve controller 104 is disposed on each of the opposite sides of temperature regulator 11. Hence, disposition of housing 20a on microchannel chip 10 causes temperature regulator 11 to be placed on metal film 200b so as to be in contact therewith. By temperature regulator 11 heating or cooling metal film 200b executes a PCR process on liquid 50 retained in microchannel 202 of microchannel chip 10. Thus, DNA can be amplified.
  • temperature regulator 11 heats or cools treatment target liquid 50 in microchannel 202 of microchannel chip 10.
  • Exemplary temperature regulator 11 is a Peltier element.
  • Temperature regulator 11 is electrically connected to heating/cooling controller 13 and power source 12 via electrical lines. Power source 12 applies voltage to temperature regulator 11.
  • Heating/cooling controller 13 refers, for example, to the relationship information among the type of liquid 50, a predetermined temperature, a period of applying the voltage, the voltage to temperature regulator 11 or the standard thereof, and controls the temperature of temperature regulator 11 by controlling the voltage application of power source 12.
  • the predetermined relationship information or the standard is the relationship information or the standard for controlling temperatures in any known PCR method.
  • any known PCR method for example, see the document ( Joan M. Henson et. al., "POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS", Annu. Rev. Phytopathol, 1993, 31, pp. 81-109 ).
  • Valve controller 104 controls opening/closing operations of introduction-side valve 51 and discharge-side valve 52.
  • exemplary valve controller 104 is structured by first projection 104a and second projection 104b that are fixed on the opposite sides of temperature regulator 11 at a lower portion of housing 20a of heating/cooling control apparatus 20 as being projecting downward.
  • first projection 104a presses introduction-side valve 51 downward to cause elastic deformation thereby closing introduction-side valve 51
  • second projection 104b presses discharge-side valve 52 downward to cause elastic deformation thereby closing discharge-side valve 52.
  • first projection 104a and second projection 104b i.e., close position II
  • introduction-side valve 51 and discharge-side valve 52 are kept in the closed state by first projection 104a and second projection 104b (i.e., close position II).
  • introduction-side valve 51 and discharge-side valve 52 are kept in the open state by being released from the pressing of first projection 104a and second projection 104b (i.e., open position I).
  • the raising/lowering operations of housing 20a integrally with temperature regulator 11 and valve controller 104 can be realized through use of any known raising/lowering apparatus such as a linear-motion mechanism made up of a motor and a ball screw shaft or an air cylinder.
  • valve controller 104 simultaneously closes introduction-side valve 51 and discharge-side valve 52.
  • valve controller 104 may be in other exemplary structure shown in FIGS. 2D and 2E , in place of that shown in FIG. 2C .
  • This other exemplary valve controller 104 is structured by first and second actuators 104c, 104d made of shape memory alloy that is capable of vertically expanding and contracting, and actuator controller 104e that controls expansion and contraction of each of first and second actuators 104c, 104d.
  • Use of shape memory alloy as first and second actuators 104c, 104d provides excellent responsiveness, and also advantageous in miniaturizing microchannel system 1 because great drive force is not required.
  • first and second actuators 104c, 104d are driven or stopped, whereby expansion/contraction operations of each of first and second actuators 104c, 104d are controlled.
  • the expansion/contraction operations of first and second actuators 104c, 104d control opening/closing operations of introduction-side valve 51 and discharge-side valve 52.
  • first and second actuators 104c, 104d are caused to expand so as to press downward introduction-side valve 51 and discharge-side valve 52 against the biasing force of supporters 51b, 52b.
  • introduction-side valve 51 and discharge-side valve 52 are deformed so as to lower from open position I to close position II against the biasing force of supporters 51b, 52b.
  • first and second actuators 104c, 104d are caused to contract so as to extinguish the pressing force on introduction-side valve 51 and discharge-side valve 52.
  • introduction-side valve 51 and discharge-side valve 52 are pushed upward by the biasing force of supporters 51b, 52b, and rise from close position II to open position I.
  • Heating/cooling controller 13 and actuator controller 104e may be implemented by one or more electronic circuit that includes a semiconductor apparatus, a semiconductor integrated circuit (i.e., IC), or an LSI (i.e., large scale integration).
  • the LSI or the IC may be integrated on one chip, or may be structured by a combination of a plurality of chips. For example, functional blocks other than a memory element may be integrated on one chip.
  • microchannel system 1 is prepared. That is, under housing 20a of heating/cooling control apparatus 20, microchannel chip 10 shown in FIG. 3A is installed. Specifically, above the upper surface of second substrate layer 200 (that is, metal film 200b), temperature regulator 11 that is fixed to housing 20a of heating/cooling control apparatus 20 is disposed. Here, introduction-side valve 51 and discharge-side valve 52 are positioned at open position I.
  • valve controller 104 is structured by first projection 104a and second projection 104b, as described above, in Step S001, temperature regulator 11 is positioned so as to be spaced apart above from the upper surface of second substrate layer 200 (i.e., metal film 200b).
  • valve controller 104 is structured by first and second actuators 104c, 104d and actuator controller 104e, as shown in FIG. 3B , temperature regulator 11 is placed on the upper surface of second substrate layer 200 (i.e., metal film 200b) together with housing 20a.
  • actuator controller 104e does not drive first and second actuators 104c, 104d and valves 51, 52 are kept at open position I.
  • treatment target liquid 50 is introduced into introducing channel 101 from introducing opening 101c at first substrate layer 100.
  • introduction-side valve 51 and discharge-side valve 52 are at open position I by being opened by valve controller 104, liquid 50 is introduced into introducing channel 101.
  • introduction-side valve 51 and discharge-side valve 52 are closed.
  • treatment target liquid 50 is packed in microchannel 202.
  • valves 51, 52 may be controlled to close through use of a sensor (not shown) disposed at discharging opening 102c sensing that treatment target liquid 50 is discharged from discharging opening 102c.
  • treatment target liquid 50 may be packed in microchannel 202 by: previously obtaining the volume of liquid 50 that can be packed in introducing channel 101, microchannel 202, and discharging channel 102; introducing liquid 50 of the obtained volume from introducing channel 101; and thereafter closing introduction-side valve 51 and discharge-side valve 52 by valve controller 104.
  • valve controller 104 may exert control to open or close introduction-side and discharge-side valves 51, 52 referring to the standard defining the volume of introduced liquid 50 and the period from when the liquid introduction is started until when introduction-side and discharge-side valves 51, 52 are closed, based on information including the viscosity of liquid 50, the introduction amount of liquid 50, and the capacity of introducing channel 101, microchannel 202, and discharging channel 102.
  • valve controller 104 is structured by first projection 104a and second projection 104b
  • introduction-side and discharge-side valves 51, 52 can be controlled to close by causing housing 20a having valve controller 104 to be placed so as to be in contact with the upper surface of second substrate layer 200.
  • valve controller 104 is structured by first and second actuators 104c, 104d and actuator controller 104e
  • introduction-side and discharge-side valves 51, 52 can be positioned at close position II by actuator controller 104e driving first and second actuators 104c, 104d.
  • heating/cooling controller 13 refers to PCR process information such as a predetermined standard, and exerts control using power source 12 to raise or lower the temperature of temperature regulator 11 that is placed so as to be in contact with metal film 200b at second substrate layer 200.
  • the heat of temperature regulator 11 is conducted to treatment target liquid 50 in microchannel 202 via the upper surface of second substrate layer 200 (i.e., metal film 200b).
  • a PCR process is executed on treatment target liquid 50 in microchannel 202.
  • an exemplary PCR process repeats, for 30 cycles, a series of processes, namely, an annealing process at 95°C for 30 seconds, a denaturation process at 72°C for 30 seconds, and an extension process at 60°C for 30 seconds.
  • valve controller 104 exerts control to open introduction-side and discharge-side valves 51, 52, to be positioned at open position I. Thereafter, from introducing opening 101c at first substrate layer 100, pure water or gas such as air is supplied into introducing channel 101, to discharge liquid 50 from introducing channel 101, microchannel 202, and discharging channel 102.
  • pure water or gas such as air is supplied into introducing channel 101, to discharge liquid 50 from introducing channel 101, microchannel 202, and discharging channel 102.
  • the upper surface of microchannel 202 that retains treatment target liquid 50 is structured by metal film 200b, which is greater in thermal conductivity than second substrate layer 200 excluding the upper surface, and metal film 200b and liquid 50 are allowed to be in direct contact with each other. This makes it possible to reduce diffusion of heat of temperature regulator 11 via metal film 200b, and to improve responsiveness of the temperature of treatment target liquid 50 by temperature regulator 11.
  • metal film 200b is circular as shown in FIG. 4A .
  • metal film 200b may conform to the shape of microchannel 202 as shown in FIGS. 4B and 4C .
  • metal film 250 is also S-shaped. In this manner, any portion of metal film 250 not in contact with liquid 50 can be eliminated, and heat from temperature regulator 11 can be more efficiently conducted to liquid 50.
  • metal film 200b, 250 is not limited to a single-film structure as shown in FIG. 5A .
  • the metal film may be a plurality of small circular metal films 251 that are arranged spaced apart from each other along S-shaped microchannel 202. It goes without saying that the present disclosure is not limited to the S-shaped arrangement, and a multitude of small circular metal films may be provided in a circular shape as shown in FIGS. 4B and 4C .
  • substrate layer body 200a of synthetic resin-made second substrate layer 200 exists between dotty metal films 251
  • the elastic force of substrate layer body 200a allows the surface where metal films 251 are arranged to become uneven so as to conform to unevenness 11a of the surface of the Peltier element, which is exemplary temperature regulator 11.
  • metal films 251 can be closely in contact with unevenness 11a of the surface of the Peltier element.
  • Metal films 251 are not limited to be dotted in a circular shape, and may be in any shape such as elliptical.
  • a specific structure of metal film 200b in microchannel 202 for preventing leakage of the liquid may be as follows.
  • FIGS. 6A to 6E are vertical cross-sectional views for describing an exemplary process of molding microchannel 202 and metal film 200b.
  • the figures show the case where a portion of microchannel 202 and metal film 200b are integrally molded.
  • the upper surface of microchannel 202 is structured by metal film 200b, it is important to prevent leakage of liquid 50 from between substrate layer body 200a and metal film 200b.
  • liquid 50 can be prevented from leaking.
  • FIG. 6A shows part of mold assembly 300 for second substrate layer 200 for molding a portion of microchannel 202.
  • Mold assembly 300 has approximately C-shaped cavity 300a for forming microchannel 202.
  • FIG. 6B shows the state where metal film 200b is disposed at the upper surface in cavity 300a.
  • FIG. 6C shows the state where synthetic resin 301, e.g., PDMS, for substrate layer body 200a of second substrate layer 200 is packed by injection molding into cavity 300a shown in FIG. 6B .
  • synthetic resin 301 e.g., PDMS
  • FIG. 6D shows the state where a mold product of a "PCR component” is taken out from cavity 300a.
  • the mold product is made up of substrate layer body 200a and metal film 200b integrally fixed to each other.
  • tails 302 of synthetic resin are formed. That is, metal film 200b made of high-thermal-conductivity metal such as aluminum is previously placed in cavity 300a, and thereafter synthetic resin 301 such as PDMS is injected into cavity 300a, to form the "PCR component" of a portion of microchannel 202.
  • synthetic resin 301 such as PDMS
  • FIG. 6E is an enlarged vertical cross-sectional view of tail 302.
  • Peltier element is used as exemplary temperature regulator 11 in the exemplary embodiment, a heat exchanger that causes hot water and cold water to flow to heat or cool the liquid may be used in place of the Peltier element.
  • valves 51, 52 are fixed to housing 20a of heating/cooling control apparatus 20, the disposition of Peltier element 11 so as to be in contact with metal film 200b and a driving operation of valves 51, 52 are associated with each other.
  • the disposition of Peltier element 11 so as to be in contact with metal film 200b and a driving operation of valves 51, 52 are associated with each other.
  • valves 51, 52 can be raised or lowered without being associated with the disposition.
  • any appropriate combination of the various exemplary embodiments and Variations can achieve their respective effects. Further, a combination of exemplary embodiments, a combination of Examples, or a combination of an exemplary embodiment and Example is also effective. Further, a combination of characteristics in different exemplary embodiments or Examples is also effective.
  • the microchannel chip, the PCR method, and the heating/cooling control apparatus reduces diffusion of heat of the temperature regulator, and the temperature regulator is capable of improving responsiveness of the temperature of the treatment target liquid. Accordingly, they are useful as a microchannel chip a PCR method, and a heating/cooling control apparatus with each of which a PCR method for sensing a DNA is performed.

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Claims (10)

  1. Polymerase-Kettenreaktions-(PCR)-Verfahren zur Durchführung eines PCR-Prozesses an einer Behandlungszielflüssigkeit (50) unter Verwendung einer Mikrokanalvorrichtung (1), die einen Mikrokanalchip (10) und eine Heiz-/ Kühlsteuervorrichtung (20) enthält, wobei das PCR-Verfahren umfasst:
    (a) Vorbereiten der Mikrokanalvorrichtung (1), die den Mikrokanalchip (10) und die Heiz-/Kühlsteuervorrichtung (20) enthält; wobei
    der Mikrokanalchip (10) umfasst:
    eine erste Substratschicht (100) mit einem Einleitkanal (101) und einem Auslasskanal (102); und
    eine zweite Substratschicht (200), die auf der ersten Substratschicht (100) angeordnet ist und einen zweiten Substratschichtaufbau (200a) und eine Metallfolie (200b) aufweist, wobei der zweite Substratschichtaufbau (200a) einen Mikrokanal (202) einschließt, der mit dem Einleitkanal (101) und dem Auslasskanal (102) verbunden ist, der Mikrokanal (202) mit der Behandlungszielflüssigkeit (50) gefüllt ist und die Metallfolie (200b) eine Oberfläche des Mikrokanals (202) strukturiert,
    die Heiz-/Kühlsteuervorrichtung (20) enthält:
    einen Temperaturregler (11), der so angeordnet ist, dass er sich mit der Metallfolie (200b) der zweiten Substratschicht (200) in Kontakt befindet und in der Lage ist, die Metallfolie (200b) zu erhitzen oder zu kühlen;
    eine Stromquelle (12), die an den Temperaturregler (11) Spannung anlegt; und
    eine Heiz-/Kühlsteuereinheit (13), welche die von der Stromquelle (12) an den Temperaturregler (11) angelegte Spannung steuert;
    (b) Steuern, mit der Heiz-/Kühlsteuereinheit (13), die von der Stromquelle (12) an den Temperaturregler (11) angelegte Spannung, um eine Temperatur der Behandlungszielflüssigkeit (50) in dem Mikrokanal (202) zu ändern, wodurch der PCR-Prozess erreicht wird,
    wobei ein Ende der Metallfolie (200b) der zweiten Substratschicht (200) im Mikrokanalchip in dem zweiten Substratschichtaufbau (200a) vergraben wird.
  2. PCR-Verfahren nach Anspruch 1, wobei
    der Mikrokanalchip (10) des Weiteren enthält:
    ein einleitungsseitiges Ventil (51), das in der Lage ist, den Einleitkanal (101) zu öffnen oder zu schließen; und
    ein auslassseitiges Ventil (52), das in der Lage ist, den Auslasskanal (102) zu öffnen oder zu schließen,
    wobei die Heiz-/Kühlsteuervorrichtung (20) des Weiteren enthält:
    eine Ventilsteuereinheit, die das einleitungsseitige Ventil (51) und das auslassseitige Ventil (52) zum Öffnen oder Schließen steuert,
    wobei das PCR-Verfahren des Weiteren vor dem Schritt (b)
    (c) Schließen des einleitungsseitigen Ventils (51) und des auslassseitigen Ventils (52) unter Steuerung der Ventilsteuereinheit in einem Zustand, bei dem der Mikrokanal (202) mit der Behandlungszielflüssigkeit (50) gefüllt ist, umfasst.
  3. PCR-Verfahren nach Anspruch 1, wobei der zweite Substratschichtaufbau (200a) aus Dimethylpolysiloxan besteht und die Metallfolie (200b) aus Aluminium besteht.
  4. PCR-Verfahren nach Anspruch 1, wobei der Temperaturregler (11) ein Peltier-Element ist.
  5. PCR-Verfahren nach Anspruch 1, wobei die Metallfolie (200b) eine Vielzahl von Metallfolien enthält, die im Abstand voneinander und längs des Mikrokanals (202) angeordnet sind.
  6. PCR-Verfahren nach Anspruch 2, des Weiteren umfassend, nach dem Schritt (b),
    (d) Öffnen des einleitungsseitigen Ventils (51) und des auslassseitigen Ventils (52), um aus dem auslassseitigen Ventil (52) die Behandlungszielflüssigkeit (50), nachdem sie den PCR-Prozess durchgemacht hat, zu entleeren.
  7. PCR-Verfahren nach Anspruch 1, wobei im Schritt (b) die Heiz-/Kühlsteuervorrichtung (20) die Temperaturänderung des Temperaturreglers (11) entsprechend einem Typ der Behandlungszielflüssigkeit (50) steuert, basierend auf Beziehungsinformationen zwischen dem Typ der Behandlungszielflüssigkeit (50), der Spannung an den Temperaturregler (11), einer Zeitdauer zum Anlegen der Spannung und der Temperatur.
  8. Mikrokanalvorrichtung, die zur Durchführung eines Polymerase-Kettenreaktions-(PCR)-Prozesses an einer Behandlungszielflüssigkeit (50) verwendet wird, wobei die Mikrokanalvorrichtung einen Mikrokanalchip (10) und eine Heiz-/Kühlsteuervorrichtung (20) enthält,
    wobei der Mikrokanalchip (10) umfasst:
    eine erste Substratschicht (100), die einen Einleitkanal (101) und einen Auslasskanal (102) enthält; und
    eine zweite Substratschicht (200), die auf der ersten Substratschicht (100) angeordnet ist und einen zweiten Substratschichtaufbau (200a) und eine Metallfolie (200b) einschließt,
    wobei der zweite Substratschichtaufbau (200a) mit dem Einleitkanal (101) und dem Auslasskanal (102) verbunden ist und einen Mikrokanal (202) enthält, um mit der Behandlungszielflüssigkeit (50) gefüllt zu werden,
    die Metallfolie (200b) eine obere Fläche des Mikrokanals (202) strukturiert, und
    die Heiz-/Kühlsteuervorrichtung (20) umfasst:
    einen Temperaturregler (11), der so angeordnet ist, dass er sich mit der Metallfolie (200b) der zweiten Substratschicht (200) in Kontakt befindet und in der Lage ist, die Metallfolie (200b) zu erhitzen oder zu kühlen;
    eine Stromquelle (12), die an den Temperaturregler (11) Spannung anlegt; und eine Heiz-/Kühlsteuereinheit (13), welche die von der Stromquelle (12) an den Temperaturregler (11) angelegte Spannung steuert;
    in einem Zustand, bei dem der Temperaturregler (11) sich mit der Metallfolie (200b) in Kontakt befindet, wobei die Heiz-/Kühlsteuereinheit (13) ausgelegt ist, die von der Stromquelle (12) an den Temperaturregler (11) angelegte Spannung zu steuern, um eine Temperatur der Behandlungszielflüssigkeit (50) zu ändern, die vom Einleitkanal (101) eingeleitet wurde, so dass der Mikrokanal (202) mit der Behandlungszielflüssigkeit (50) gefüllt ist, wodurch der PCR-Prozess erreicht wird,
    wobei ein Ende der Metallfolie (200b) der zweiten Substratschicht (200) im Mikrokanalchip in dem zweiten Substratschichtaufbau (200a) vergraben wird.
  9. Mikrokanalvorrichtung nach Anspruch 8, wobei
    der Mikrokanalchip (10) des Weiteren enthält:
    ein einleitungsseitiges Ventil (51), das in der Lage ist, den Einleitkanal (101) zu öffnen oder zu schließen; und
    ein auslassseitiges Ventil (52), das in der Lage ist, den Auslasskanal (102) zu öffnen oder zu schließen.
  10. Mikrokanalvorrichtung nach Anspruch 8, wobei der zweite Substratschichtaufbau (200a) aus Dimethylpolysiloxan und die Metallfolie (200b) aus Aluminium besteht.
EP16170761.7A 2015-07-28 2016-05-23 Mikrokanalvorrichtung und pcr-verfahren Not-in-force EP3124118B1 (de)

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EP3633366B1 (de) * 2017-04-26 2025-11-19 Shimadzu Corporation Flüssigkeitszufuhrvorrichtung und flüssigchromatograph
JP2018191608A (ja) * 2017-05-19 2018-12-06 パナソニックＩｐマネジメント株式会社 核酸増幅装置及び核酸増幅方法
US12383902B2 (en) 2018-02-07 2025-08-12 Jaydeep BHATTACHARYA Low cost microfluidic device for DNA/RNA isolation, purification and amplification using chip based PCR/RT-PCR for biosensing applications
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US10660199B1 (en) * 2018-10-30 2020-05-19 Amazon Technologies, Inc. Microfluidic channels and pumps for active cooling of circuit boards
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CN111346685B (zh) * 2020-03-10 2022-01-25 中国科学院苏州生物医学工程技术研究所 可实现快速温控的装置和方法

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